Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina

Ronald Guillermo Peláez, Carlos Enrrique Muskus, Patricia Cuervo, Marcel Marín-Villa, .

Resumen

Introducción. Los mecanismos de resistencia al antimonio pentavalente conocidos hasta el momento,se han descrito ampliamente en cepas del subgénero Leishmania, pero poco se sabe sobre lasproteínas involucradas en los mecanismos de resistencia presentes en cepas del subgénero Viannia,como Leishmania panamensis.

Objetivo. Identificar proteínas diferencialmente expresadas entre las cepas de L. panamensis(UA140), sensible y resistente al antimonio pentavalente, y analizar el posible papel de estas proteínasen mecanismos de resistencia.

Materiales y métodos. Las proteínas de las cepas, sensible y resistente al antimonio pentavalente, secompararon usando electroforesis bidimensional. Las proteínas con aumento de la expresión fueronaisladas e identificadas por espectrometría de masas mediante MALDI-TOF/TOF (Matrix AssistedLaser Desorption Ionization/Time of Flight). La expresión del ARNm de cinco de estas proteínas secuantificó mediante PCR en tiempo real.

Resultados. Los geles bidimensionales de las cepas sensible y resistente detectaron 532±39 y 541±43manchas proteicas. Se encontraron 10 manchas con aumento de la expresión en la cepa resistente,identificadas como proteínas de choque térmico (Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica),isomerasa de disulfuro, proteasa de cisteína, enolasa, factor de elongación 5-α, la subunidad 5-α delproteasoma y dos proteínas hipotéticas nombradas como Sp(2) y Sp(25).

Conclusión. Este es el primer estudio llevado a cabo con una cepa resistente al antimonio pentavalenteen L. panamensis, en el cual se han identificado proteínas que están relacionadas con el mecanismode resistencia del parásito frente al medicamento, abriendo el camino para futuros estudios de estasproteínas como blancos terapéuticos.

 

doi: http://dx.doi.org/10.7705/biomedica.v32i3.392

 

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  • Ronald Guillermo Peláez Programa de Estudio y Control de Enfermedades Tropicales, PECET, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
  • Carlos Enrrique Muskus Programa de Estudio y Control de Enfermedades Tropicales, PECET, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
  • Patricia Cuervo Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, Brasil
  • Marcel Marín-Villa Programa de Estudio y Control de Enfermedades Tropicales, PECET, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia

Citas

WHO. Control of the leishmaniases: Report of a meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, March 22-26, 2010. WHO technical report series.

Geneva: WHO; 2010. p. 1-202.

Desjeux P. Leishmaniasis: Current situation and new perspectives. Comp Immunol Microbiol Infect Dis. 2004;27:305-18. http://dx.doi.org/10.1016/j.cimid.2004.03.004

Davies CR, Kaye P, Croft SL, Sundar S. Leishmaniasis: New approaches to disease control. BMJ. 2003;326:377-82. http://dx.doi.org/10.1136/bmj.326.7385.377

Requena JM, Iborra S, Carrion J, Alonso C, Soto M. Recent advances in vaccines for leishmaniasis. Expert Opin Biol Ther. 2004;65:1505-17. http://dx.doi.org/10.1517/14712598.4.9.1505

Rocha RA, Sampaio RN, Guerra M, Magalhaes A, Cuba CC, Barreto AC, et al. Apparent glucantime failure in five patients with mucocutaneous leishmaniasis. Am J Trop Med Hyg. 1980;83:131-9.

El-Masum MA, Evans DA. Characterization of Leishmania isolated from patients with Kala-azar and post Kala-azar dermal leishmaniasis in Bangladesh. Trans R Soc Trop Med

Hyg. 1995;89:331-2.

Bonfante GR, Barroeta S, Mejía de los Alejos MA, Meléndez E, Torrealba J, Valdivia O, et al. Disseminated American cutaneous leishmaniasis. Int J Dermatol. 1996;35:561-5.

Costa JM, Marsden PM, Llanos-Cuentas EA, Netto EM, Carvalho EM, Barral A, et al. Disseminated cutaneous leishmaniasis in a field clinic in Bahia, Brazil: A report of eight cases. J Trop Med Hyg. 1986;89:319-23.

Ribeiro RN, Marsden PD. Mucosal leishmaniasis unresponsive to glucantime therapy successfully treated with AmBisomeTM. Trans R Soc Trop Med Hyg. 1997;91:77.

Vélez I, Agudelo S, Hendrickx E, Puerta J, Grogl M, Modabber F, et al. Inefficacy of allopurinol as monotherapy for Colombian cutaneous leishmaniasis: A randomized, controlled trial. Ann Intern Med. 1997;126:232-6.

Palacios R, Osorio LE, Grajalew LF, Ochoa MT. Treatment failure in children in a randomized clinical trial with 10 and 20 days of meglumine antimonate for cutaneous leishmaniasis due to Leishmania Viannia species. Am J Trop Med Hyg. 2001;64:187-93.

Rojas R, Valderrama L, Valderrama M, Varona MX, Ouellette M, Saravia NG. Resistance to antimony and treatment failure in human Leishmania (Viannia) infection. J Infect Dis. 2006;193:1375-83. http://dx.doi.org/10.1086/503371

Faraut-Gambarelli F, Piarroux R, Deniau M, Giusiano B, Marty P, Michel G, et al. In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: A study of

strains collected from patients with visceral leishmaniasis. Antimicrob Agents Chemother. 1997;41:827-30.

Lira R, Sundar S, Makharia A, Kenney R, Gam A, Saraiva E, et al. Evidence that the high incidence of treatment failures in Indian Kala-azar is due to the emergence of antimony-resistant strains of Leishmania donovani. J Infect Dis. 1999;180:564-7. http://dx.doi.org/10.1086/314896

Moreira ES, Guerra JB, Petrilló-Peixoto M de L. Glucantime resistant Leishmania promastigotes are sensitive to pentostam. Rev Soc Bras Med Trop. 1992;25:247-50.

Grogl M, Thomason TN, Franke ED. Drug resistance in leishmaniasis: Its implication in systemic chemotherapy of cutaneous and mucocutaneous disease. Am J Trop Med Hyg. 1992;47:117-26.

Sereno D, Holzmuller P, Mangot I, Cuny G, Ouaissi A, Lemesre J. Antimonial-mediated DNA fragmentation in Leishmania infantum amastigotes. Antimicrob Agents Chemother. 2001;45:2064-9. http://dx.doi.org/10.1128/AAC.45.7.2064-2069.2001

Croft SL, Coombs GH. Leishmaniasis –current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol. 2003;19:502-8. http://dx.doi.org/10.1016/j.pt.2003.09.008

Wyllie S, Cunningham ML, Fairlamb AH. Dual action of antimonial drugs on thiol redox metabolism in the human pathogen Leishmania donovani. J Biol Chem. 2004;279:39925-32. http://dx.doi.org/10.1074/jbc.M405635200

Berman JD, Waddel D, Hanson BD. Biochemical mechanisms of the antileishmanial activity of sodium stibogluconate. Antimicrob Agents Chemother. 1985;27:916-20.

Berman JD, Gallalee JV, Best JM. Sodium stibogluconate (Pentostam) inhibition of glucose catabolism via the glycolytic pathway and fatty acid beta-oxidation in Leishmania

mexicana amastigotes. Biochem Pharmacol. 1987;36:197-201. http://dx.doi.org/10.1016/0006-2952(87)90689-7

Tovar J, Cunningham ML, Smith AC, Croft SL, Fairlamb AH. Down-regulation of Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant

homologue: Effect on parasite intracellular survival. Proc Natl Acad Sci. 1998;95:5311-6.

Croft SL, Sundar S, Fairlamb AH. Drug resistance in leishmaniasis. Clin Microbiol Rev. 2006;19:111-26. http://dx.doi.org/10.1128/CMR.19.1.111-126.2006

Muñoz DL, Cardona DP, Cardona A, Carrillo LM, Quiñones W, Echeverri F, et al. Effect of hydrazones against intracellular amastigotes of Leishmania panamensis and a parasitic cystein protease. Vitae. 2006;12:5-12.

Cuervo P, Batista de Jesus J, Junqueira M, Mendonça-Lima L, Gonzalez LJ, Betancourt L, et al. Proteome analysis of Leishmania (Viannia) braziliensis by twodimensional gel electrophoresis and mass spectrometry. Mol Biochem Parasitol. 2007;154:6-21. http://dx.doi.org/10.1016/j.molbiopara.2007.03.013

Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988;9:255-62. http://dx.doi.org/10.1002/elps.1150090603

Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research. 2001;29:2002-7.

Sundar S. Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health. 2001;6:849-54. http://dx.doi.org/10.1046/j.1365-3156.2001.00778.x

Grondin K, Roy G, Ouellette M. Formation of extrachromosomal circular amplicons with direct or inverted duplications in drug-resistant Leishmania tarentolae. Mol Cell Biol. 1996;16:3587-95.

Haimeur A, Ouellete M. Gene amplification in Leishmania tarentolae selected for resistance to sodium stibogluconate. Antimicrob Agent Chemoter. 1998;42:1689-94.

Haimeur A, Brochu C, Genest P, Papadopoulou B, Ouellette M. Amplification of the ABC transporter gene PGPA and increased trypanothione levels in potassium antimonyl tartrate (SbIII) resistant Leishmania tarentolae. Mol Biochem Parasitol. 2000;108:131-5. http://dx.doi.org/10.1016/S0166-6851(00)00187-0

Kumar A, Sisodia B, Misra P, Sundar S, Shasany AK, Dube A. Proteome mapping of overexpressed membraneenriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani. Br J Clin Pharmacol. 2010;70:609-17. http://dx.doi.org/10.1111/j.1365-2125.2010.03716.x

Lee N, Bertholet S, Debrabant A, Muller J, Duncan R, Nakhasi H. Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ. 2002;9:53-64. http://dx.doi.org/10.1038/sj/cdd/4400952

Sudhandiran G, Shaha C. Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. J Biol Chem. 2003;278:25120-32. http://dx.doi.org/10.1074/jbc.M301975200

Arnoult D, Akarid K, Grodet A, Petit PX, Estaquier J, Ameisen, JC. On the evolution of programmed cell death: Apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization. Cell Death Differ. 2002;9:65-81. http://dx.doi.org/10.1038/sj/cdd/4400951

Zangger H, Mottram JC, Fasel N. Cell death in Leishmania induced by stress and differentiation: Programmed cell death or necrosis? Cell Death Diff. 2002;9:1126-39. http://dx.doi.org/10.1038/sj.cdd.4401071

Brochu C, Haimeur A, Ouellette M. The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite Leishmania. Cell Stress Chaperones. 2004;9:294-303. http://dx.doi.org/10.1379/CSC-15R1.1

Baptiste V, Benjamin G, Isabelle G, Sundar S, Drummelsmith J, Ouellette M. A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death. Mol Cell Proteomics. 2007;6:88-101. http://dx.doi.org/10.1074/mcp.M600319-MCP200

Wardleworth BN, Russell RJ, Bell SD, Taylor GL, White MF. Structure of Alba: An archaeal chromatin protein modulated by acetylation. EMBO J. 2002;21:4654-62. http:// dx.doi.org/10.1093/emboj/cdf465

Bell SD, Botting CH, Wardleworth BN, Jackson SP, White MF. The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation. Science. 2002;296:148-51. http://dx.doi.org/10.1126/science.1070506

Halliwell B, Gutteridge JM. Free radicals in biology and medicine. Third edition. Oxford: Oxford University Press; 1999. p. 1-899.

Cómo citar
Peláez, R. G., Muskus, C. E., Cuervo, P., & Marín-Villa, M. (2012). Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina. Biomédica, 32(3), 418-29. https://doi.org/10.7705/biomedica.v32i3.392
Publicado
2012-05-02
Sección
Artículos originales